Andrea Petrucca

Multiple-Antibiotic Resistance Mediated by Structurally Related IncL/M Plasmids Carrying an Extended-Spectrum β-Lactamase Gene and a Class 1 Integron

ABSTRACT A conjugative IncL/M plasmid (pSEM) conferring resistance to gentamicin, amikacin, kanamycin, sulfonamides, and expanded-spectrum cephalosporins was found in pathogenic strains of Salmonella enterica serotype Typhimurium. Resistance to aminoglycosides was encoded by a sul1-type class 1 integron (In-t3). An extended-spectrum beta-lactamase gene,blaSHV-5, was identified 3.5 kb downstream of the integrase (intI1) gene of In-t3. Nucleotide sequence analysis of the 5.3-kb blaSHV-5–In-t3 region of pSEM highlighted striking similarities with IncL/M plasmids isolated from nosocomial gram-negative pathogens, conferring resistance to expanded-spectrum cephalosporins and aminoglycosides.

Antibody Response to Chlamydial Heat Shock Protein 60 Is Strongly Associated With Acute Coronary Syndromes

Background Heat shock proteins (HSPs) are a family of proteins with immunogenic and proinflammatory properties. Human and Chlamydia pneumoniae (Cp) HSP60 were found in patients with stable coronary disease. Methods and Results We measured the levels of anti‐Cp‐HSP60 and anti‐Cp immunoglobulin G (IgG) in 179 patients with unstable angina, 40 with acute myocardial infarction, and 40 with stable angina (SA), as well as 100 control subjects. Forty‐one patients with acute coronary syndromes (ACS) were also studied at follow‐up. We also measured plasma levels of high‐sensitivity C‐reactive protein (hs‐CRP) and troponin T (TnT). Seropositivity to Cp‐HSP60 was found in 99% of ACS patients but in only 20% of SA patients and none of the control subjects. Seropositivity to Cp was detected in 67% of ACS patients, 60% of SA patients, and 30% of the control subjects. No differences in Cp‐HSP60 IgG and in Cp IgG were observed between patients with myocardial infarction and patients with unstable angina. No correlation was found between Cp‐HSP60 IgG, TnT, and hs‐CRP or between IgG against Cp and hs‐CRP. In ACS patients at follow‐up, Cp‐HSP60 IgG decreased from 0.88±0.25 to 0.45±0.14 arbitrary units (P<0.0001), becoming negative in 12 patients. Conclusions Seropositivity for Cp‐HSP60 appears to be a very sensitive and specific marker of ACS, unrelated to Cp IgG antibody titers or hs‐CRP and TnT levels. Its causal involvement in instability and its diagnostic role in ACS deserve further study. (Circulation. 2003;107:3015‐3017.)

Changing carbapenemase gene pattern in an epidemic multidrug-resistant Acinetobacter baumannii lineage causing multiple outbreaks in central Italy

OBJECTIVES infections caused by multidrug-resistant (MDR) Acinetobacter baumannii are a challenging problem worldwide. Here, the molecular epidemiology and the genetic basis of antibiotic resistance in 111 MDR A. baumannii strains isolated from June 2005 to March 2009 from infected patients in 10 intensive care units (ICUs) in central Italy were investigated. METHODS epidemiological typing was performed by random amplification of polymorphic DNA, PCR-based sequence grouping and macrorestriction analysis. MICs of antibiotics were determined by the broth microdilution method. Genes for OXA carbapenemases, metallo-β-lactamases and the CarO porin were searched for by PCR. RESULTS molecular genotyping identified one predominant A. baumannii lineage, related to the international clonal lineage II, accounting for 95.6% of isolates. Isolates referable to this lineage were recovered from all ICUs surveyed and were resistant to nearly all classes of antimicrobials, with the exception of tigecycline and colistin. A high percentage (60.5%) of A. baumannii isolates showed elevated resistance to imipenem (MICs ≥  128 mg/L), concomitant with resistance to meropenem. Carbapenem resistance was associated with the presence of either bla(OXA-58)-like (22.8%) or bla(OXA-23)-like (71.1%) carbapenemase genes. Molecular typing showed that the epidemic lineage encoding OXA-23 emerged in 2007 and displaced a genetically related clone encoding OXA-58 that had been responsible for previous ICU outbreaks in the same region. CONCLUSIONS emergence of the OXA-23 epidemic lineage could result from selective advantage conferred by the bla(OXA-23)-like determinant, which provides increased resistance to carbapenems.

Antibodies to 60-Kilodalton Heat Shock Protein and Outer Membrane Protein 2 of Chlamydia pneumoniae in Patients with Coronary Heart Disease

ABSTRACT Evidence linking Chlamydia pneumoniae infection to atherosclerosis and to atherothrombotic events has recently emerged. A primary candidate implicated in these pathogenetic events is the 60-kDa chlamydial heat shock protein (HSP60). Another putative candidate to activate a potential proinflammatory mechanism is the chlamydial outer membrane protein 2 (OMP2). We have generated both HSP60 and OMP2 recombinant antigens in a nondenatured form and shown that (i) the two antigens were highly immunogenic in mice and (ii) murine antisera thus generated recognized the native C. pneumoniae proteins. We measured by enzyme linked immunosorbent assay (ELISA) and immunoblot assay antibody titers to the recombinant antigens in samples from 219 patients with coronary heart disease (CHD), 179 patients with unstable angina (UA), 40 patients with acute myocardial infarction (AMI), and 100 age-, sex-, and risk factor-matched healthy controls. We also examined whether anti-HSP60 and/or anti-OMP2 antibodies correlated with anti-C. pneumoniae antibodies assessed by a commercial microimmunofluorescence (MIF) assay. Immunoglobulin G (IgG), but neither IgA nor IgM, antibodies against the two recombinant proteins were detected by ELISA. In particular, anti-HSP60 antibodies were detected in >99% of CHD patients versus 0% of the controls, whereas the proportions of anti-OMP2 positive subjects were >70 and 27%, respectively. Nonetheless, among CHD patients, similar frequencies of positive subjects and titers of anti-HSP60 or anti-OMP2 antibodies were present in UA and AMI subjects. The anti-OMP2, but not the anti-HSP60, antibodies showed high specificity. Consistently, high serological correlation was observed between IgG MIF titers and IgG ELISA reactivity to OMP2 but not to HSP60. Overall, the results of this study demonstrate a strong correlation between CHD and anti-HSP60 IgG levels, as measured by our in-house ELISA. They also suggest that recombinant OMP2 ELISA, because of its high specificity and strong correlation with MIF assay, could be a candidate diagnostic marker for C. pneumoniae infection, which would be of potential usefulness for its specificity and nonsubjective nature.

The truncated hemoglobin from Mycobacterium leprae

Truncated hemoglobins (trHbs) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHbs branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHbs, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.

Travel-Associated Burkholderia pseudomallei Infection (Melioidosis) in a Patient with Cystic Fibrosis: A Case Report

In September 1997, a 25-year-old Italian woman with cystic fibrosis (CF) spent 3 weeks in Thailand. In August 1998, her pulmonary function rapidly declined, with productive cough and intermittent fever. Chest x-ray films revealed diffuse, small, patchy opacities in the upper lobes. Burkholderia pseudomallei (BP) was isolated from specimens of the patients sputum and was identified by means of 16S rDNA sequencing. The diagnosis of melioidosis was serologically confirmed. Continuous therapy with ceftazidime and co-trimoxazole and maintenance with co-trimoxazole, doxycycline, and chloramphenicol resulted in eradication of BP. We present the issue of whether patients with CF represent a population particularly at risk for melioidosis.

Identification and quantification of Chlamydia pneumoniae in human atherosclerotic plaques by LightCycler real-time-PCR.

A real-time PCR assay for Chlamydia pneumoniae in human atherosclerotic plaques by the use of novel probes and FRET LightCycler technology, is described. The assay proved particularly suitable for the specific and quantitative detection of a low DNA copy number in conventional PCR-negative samples. Among fifteen nested-PCR negative atherosclerotic plaques examined, our method detected three positive plaques containing 50(+/-3), 37(+/-2) and 24(+/-2) DNA copy number+/-SD in three independent experiments. Real-time PCR holds promise for C. pneumoniae quantitation in human atherosclerotic plaques.

Stenotrophomonas maltophilia strains from cystic fibrosis patients: Genomic variability and molecular characterization of some virulence determinants

The genetic relatedness of 52 Stenotrophomonas maltophilia strains, collected from various environmental and clinical sources, including cystic fibrosis (CF) patients, as well as the presence and the expression of some virulence-associated genes were studied. Pulsed-field gel electrophoresis (PFGE) analysis identified 47 profiles and three clusters of isolates with an identical PFGE pattern considered to be indistinguishable strains. Restriction fragment length polymorphism of the gyrB gene grouped the 52 strains into nine different profiles. Most CF clinical isolates (29 out of 41) showed profile 1, while the analysis of the hypervariable regions of the 16S rRNA gene revealed five distinct allelic variations, with the majority of CF isolates (23 out of 41) belonging to sequence group 1. Furthermore, the strains were characterized for motility and expression of virulence-associated genes, including genes encoding type-1 fimbriae, proteases (StmPr1 and StmPr2) and esterase. All S. maltophilia strains exhibited a very broad range of swimming and twitching motility, while none showed swarming motility. A complete smf-1 gene was PCR-amplified only from clinically derived S. maltophilia strains. Finally, the virulence of representative S. maltophilia strains impaired in the expression of proteases and esterase activities was evaluated by infecting larvae of the wax moth Galleria mellonella. The results obtained strongly indicate that the major extracellular protease StmPr1 may be a relevant virulence factor of S. maltophilia.

Enteroinvasive Escherichia coli virulence-plasmid-carried apyrase (apy) and ospB genes are organized as a bicistronic operon and are subject to differential expression.

In Shigella flexneri and enteroinvasive Escherichia coli (EIEC) the expression of the virulence-plasmid(pINV)-carried potential pathogenesis-associated apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated by the same regulators that govern the expression of virulence genes. To understand the transcriptional organization of the apy gene, the authors sequenced an 8023 bp PstI fragment of the pINV of EIEC strain HN280, which encompasses apy as well as its adjacent genes. The PstI fragment displays 99% identity with the corresponding fragment of pWR100, the pINV of S. flexneri strain M90T, and contains four genes. One of these genes, ospB, encodes a secreted protein of unknown activity and is located immediately upstream of apy. Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that ospB and apy are co-transcribed as a 2 kb bicistronic, temperature-regulated mRNA from an upstream promoter that precedes ospB. The 2 kb mRNA is post-transcriptionally processed in the intercistronic ospB-apy region, leading to the considerable accumulation of a more stable 1 kb apy-specific mRNA (half-life of 2.2+/-0.3 min, versus 27+/-4 s for the 2 kb transcript). Upon temperature induction, peak expression of the ospB-apy operon occurs when bacteria enter into the late phases of bacterial growth, where the apy-specific transcript was found to be much more prevalent if compared to the ospB-apy transcript.

Differentiation of leptospires of the serogroup Pomona by monoclonal antibodies, pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction

All reference strains described as representing separate serovars belonging to the serogroup Pomona and a clinical leptospiral isolate (LP2) from this serogroup were analyzed using a battery of 9 monoclonal antibodies, pulsed-field gel electrophoresis (PFGE) and arbitrarily primed polymerase chain reaction (AP-PCR). Monoclonal antibody analysis provided taxonomic results which were in agreement with the current classification of the serogroup Pomona into six serovars and allowed the classification of the isolate LP2 in the serovar pomona. PFGE and AP-PCR, although in general agreement with monoclonal antibody analysis, also were able to demonstrate some differences in the restriction patterns of strains Pomona, Monjakov and CB. These results indicate that these strains, grouped within serovar pomona after the introduction of bacterial restriction endonuclease analysis as the typing method, but formerly described as representing separate serovars (pomona, monjakov and cornelli, respectively), are similar but not identical to one another. This was also the case with strains 5621, the serovar mozdok reference strain, and K1, formerly described as serovar dania reference strain, but currently recognized to be a mozdok-like strain. These findings suggest that the deletion of some serovars within the serogroup Pomona, namely mozdok, cornelli, and dania, should be reconsidered. Thus, PFGE appears to be a useful tool for the serovar identification of leptospires belonging to the serogroup Pomona and for shedding light on the problem of their classification.